Summary Invariant natural killer T cells (iNKT cells) express an invariant TCR ? chain and they can recognize self- derived as well as microbial glycolipid antigens presented by CD1d. Mice lacking iNKT cells are impaired in their early immune response against Streptococcus pneumoniae, a gram-positive bacterium responsible for pneumonia, sepsis and other diseases. We have shown that S. pneumoniae synthesizes a glycolipid that is a both a major component of their membrane and an antigen for iNKT cells. This compound is a glucosylated diacylglycerol (Glc-DAG) containing vaccenic acid, a mono-unsaturated, 18 carbon fatty acid (C18:1) with a cis unsaturated bond between carbons 9 and 10. We found an identical antigen in groupBstreptococcus(GBS),a leadingcauseofmeningitisinchildren,althoughtheroleofiNKTcellsindefensefromthispathogenremains untested.Our first guiding hypothesis, supported by data that are still preliminary, is that these microbial antigens are required for iNKT cell activation by S. pneumoniae. This understandably has been a controversial issue, in light of the self-reactivity of iNKT cells. Furthermore, as a second guiding hypothesis, we propose that some types of bacteria, including S. pneumoniae and GBS, avoid iNKT cell recognition of their membrane glycolipid by turning off synthesis of vaccenic acid in their hosts and by creating a molecular chimera by incorporating host oleic acid into their membrane glycolipid. Although oleic acid only differs from vaccenic acid only in the placement of the cis unsaturated bond, the Glc-DAG antigen with oleic acid cannot be recognized by iNKT cells. In the specific aims, we combine genetics, biochemistry and immune assays to demonstrate the importance of foreign antigen biosynthesis for iNKT cell activation and host defense. In Aim 2, using bacteria grown under different conditions and strains that report on unsaturated fatty acid biosynthesis, we will explore the timing and the organ(s) under which these two gram-positive pathogens turn off vaccenic acid synthesis, and the effect this has on the iNKT cell response and host defense in different sites, including the lung, brain, reproductive tract, as well as systemic defense. In Aim 3, we will reduce synthesis of oleic acid in infected mice, to determine if increased availability of this nutrient limits the protective iNKT cell response. In Aim 4, we explore the biochemical basis for the fine specificity of recognition of glycolipids based on the placement of the fatty acid double bond, which is buried in the CD1d antigen binding groove and therefore not directly in contact with the TCR. We also will determine if the Glc-DAG antigens with oleic acid function as effective antagonists of the Glc-DAG antigen synthesized by the bacteria. The proposed experiments are based on our novel finding that the advantage due to metabolic saving when Strep bacteria take up host C18:1 fatty acid also provides an immune evasion mechanism. The results will have impact by providing insights into the protective responses to two important pathogens and their relationships with their hosts. The data will not only deliver a greater understanding of the requirements for iNKT cell activation, but also, they will elucidate a pathogen immune evasion mechanism that is tied to the availability of an important nutrient. The results may also have implications for understanding how diet and obesity impair a protective host response.